The goals of Project 0001 are to define the antigenic determinants of carcinoembryonic antigen (CEA) recognized by two highly CEA specific monoclonal antibodies, T84.66 and T84.12, and to characterize several engineered derivatives of these antibodies and their metal ion chelates produced in other portions of this Program Project. In earlier work we have proposed that CEA is a member of the Immunoglobulin gene superfamily and that the CEA molecule can be defined by a series of Variable- and Constant-like domains. This model has been partially verified by the expression and characterization of several of these domains in the HeLa cell line, including localization of the T84.66 epitope to the A3B3 domain of CEA. Further delineation of the amino acid sequence of the epitope will be performed on the A3B3 domain and chimeric A2B3 and A3B3 domains by site-directed mutagenesis on the expressed domains in HeLa cells and in a fd phage display system. Since the domains occur in pairs, A1B2, A2B2, A3B3, and each analogous subdomain has high sequence homology, we expect that we can convert non-Ab binding chimeras A2B3 and A3B2 to Ab-binding by site-directed mutagenesis and detection by domain specific monoclonal antibodies. Amino acids selected for mutagenesis in the A3B3 domain will be conservative using equivalent substitutions from homologous domains. Proper folding of the expressed domains will be verified by immunological and physical studies (circular dichroism). These studies should define the critical amino acids involved in antibody binding, verify the domain structure of CEA, and provide a basis for modeling the antibody-antigen interactions. Analysis of the engineered antibodies produced in Project 2 includes testing of immunoreactivity before and after conjugation to chelates, verification of subunit size by laser desorption time of flight mass spectrometry, and protein sequence analysis and peptide mapping by fast atom bombardment and electrospray mass spectrometry. Metal ion chelates and complexes produced in Project 4 will be verified by mass spectrometry, conjugated to engineered antibodies, and radiolabeled to verify their appropriateness for animal studies. Based on the properties of the metal ion complexes, new protocols will be developed to optimize the conjugation and radiolabeling procedures, including the use of linkers, and site specific conjugation through the carbohydrate moieties. Linkers also affect the serum stability an d tissue clearance of the radiolabeled conjugates. This project will coordinate its efforts with Core 9006 (Animal and Clinical Lab) to optimize radiolabeled conjugates for human studies, Core 9001 (Radiopharmacy) for development of radiolabeling and conjugation procedures, and Core 9003 (Large scale production) for characterization of purified products.